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. 2012 Dec 1;125(23):5758–5769. doi: 10.1242/jcs.110007

Fig. 5.

Fig. 5.

Analysis of PLCε regulation by different small GTPases. (A) PLC activity of the WT and PLCε RAm variant was analysed in COS7 cells in the absence (black) or presence of either RalAV23 (grey) or RhoAV14 (white) (left panel). PLC activity of the WT PLCε was also tested in the presence of activated forms of RhoA, RhoB, RhoC, RalA and RalB (right panels). (B) Cell extracts from WT MEFs, untreated (−) and treated with PDGF-BB for 5 min (+), were analysed by western blotting using antibodies to RalA and RalB GTPases; total protein and protein present in the “pull-down” with the GST-RalBP1-RBD (GTP-bound forms) were analysed. (C) PLC activity was measured in immortalised cell populations of Rala flox/flox and Ralb flox/flox MEFs following infection with either Ad-GFP or Ad-Cre; untreated MEFs (basal) and MEFs treated with PDGF-BB (PDGF) were analysed. *P<0.05, t-test. (D) Expression of RalA and RalB proteins was analysed in Rala flox/flox and Ralb flox/flox MEFs following infection with either Ad-GFP or Ad-Cre. (E) Forward Migration Index was determined using Dunn chambers; immortalised populations of Rala flox/flox and Ralb flox/flox MEFs, following infection with either Ad-GFP (control) or Ad-Cre, were analysed. The data are from four independent experiments, *P<0.05, ANOVA. Random migration was not affected following infection with Ad-Cre.